Unraveling biotypes of the northern house mosquito, Culex pipiens s.l. (Diptera: Culicidae): molecular differentiation and morphometric analysis

Abstract Geometric morphometrics was used to determine whether geographic isolation could explain differences in wing size and shape between and within continental (27°S to 41°S) and insular (Rapa Nui) populations of Culex pipiens s.s. Linnaeus and their biotypes (f. pipiens and f. molestus). Molecular protocols based on polymorphisms in the second intron of nuclear locus ace-2 (acetylcholinesterase-2) were used to differentiate Cx. pipiens s.s. from Cx. quinquefasciatus Say, and an assay based on polymorphisms in the flanking region of a microsatellite locus (CQ11) was used to identify biotypes. Culex pipiens f. molestus and hybrids shared larval habitats in all continental sites, while Cx. pipiens f. pipiens was found in 5 of the 10 sites. Only biotype molestus was found in Rapa Nui (Easter Island) Pipiens and molestus biotypes occur sympatrically in aboveground locations, and only molestus was found in the underground site (ME). Biotype molestus was dominant in rural locations and preferably anthropophilic. These results agree with the ecological descriptions previously reported for the biotypes of Cx. pipiens s.s. Procrustes ANOVA only showed differences in centroid size between biotypes in females and males and did not show significant differences in wing shape. However, we found significant differences among the geographic areas in the centroid size and wing shape of both females and males. Particularly, the population of Rapa Nui Island had shorter wings than the continental populations. The results highlight the effects of geographic and environmental processes on morphotypes in vector mosquitoes.


Introduction
Mosquitoes of the genus Culex are important vectors of human diseases.Culex pipiens Linnaeus and Cx.quinquefasciatus Say are considered the primary vectors of the West Nile virus (WNV), a flavivirus introduced in the United States in the 1990s (Fonseca et al. 2004).WNV spread to Central America and the Caribbean between 2001 and 2004.In South America, WNV has been reported in horses in Argentina (Morales et al. 2006) and flamingos in Colombia (Osorio et al. 2012), and there is strong evidence of ongoing WNV transmission throughout South America, including Brazil and Venezuela (Lorenz and Chiaravatolli 2022).
Culex pipiens and Cx.quinquefasciatus are both species of the Cx.pipiens complex and are found in South America.They are highly similar morphologically but can be distinguished by the shape of the male terminalia (Dobrotworsky 1967); however, the lack of physical morphological differences in females, coupled with the existence of hybrids make identification challenging (Price and Fonseca 2015).Molecular studies using mitochondrial markers have not been successful in distinguishing between members of this complex (Laurito et al. 2013).Specific protocols based on a nuclear locus developed by Smith and Fonseca (2004) allowed the differentiation of Cx. pipiens s.s.from Cx. quinquefasciatus.Polymorphisms of the CQ11 microsatellite locus (a repeated TG sequence) have been used to differentiate between 2 biotypes or ecological forms of Cx. pipiens: f. pipiens and f. molestus (Bahnck and Fonseca 2006).Studies of Cx. pipiens biotypes have revealed behavioral and physiological differences that strongly influence their vectorial capacity (Ciota and Kramer 2013).These differences include preferences in larval habitat hypogeous (biotype molestus) vs. epigeous (biotype pipiens), rural (biotype pipiens) vs. urban (biotype molestus), and preferences in adult strategies, such as urban variations in host feeding patterns (mammophilic vs. ornithophilic), gonotrophic development (autogeny vs. anautogeny), and means or presence of adult female hibernation (quiescence vs. diapause) (Farajollahi et al. 2011).Culex pipiens f. pipiens was described mainly as anautogenic, ornithophilic, heterodynamic, and eurygamous, while Cx.pipiens f. molestus has been described as autogenic, mammophilic, homodynamic, and stenogamous (Shaikevich et al. 2016, Aardema et al. 2020).
Most studies recognize these biotypes as valid entities to infer patterns of diversity and divergence (Shaikevich et al. 2016, Vogels et al. 2016, Haba and Macbride 2022), but their validity and taxonomic position remains controversial.Given the difficulty and cost of molecular analysis, and the debate surrounding the use and validity of biotypes, geometric morphometric (GM) methods have emerged as robust tools for differentiating cryptic species within the Cx.pipiens complex (Wilke et al. 2016, Simões et al. 2020).Studies of GM differentiate epidemiologically relevant mosquitoes, particularly by comparing wing shape, as vein intersections can be used as conspicuous landmarks, and are homologous between species (Lorenz et al. 2017).Wing-shaped studies have discerned taxonomic issues in the family Culicidae as differences within (Motoki et al. 2012, Gómez et al. 2014) and between species (Laurito et al. 2015, Wilke et al. 2016, Garzón et al. 2020).GM has also allowed differentiation of species in sympatric areas (Lorenz et al. 2012).Garzón et al. (2020) correctly separated females of Cx. pipiens s.s. and Cx quinquefasciatus from Argentina using 17 landmarks and the siphonal index.These authors described that Culex quinquefasciatus had thinner wings than Cx.pipiens, which may indicate ecological adaptations to the environment in its distribution.Wu et al. (2014) found that Cx. pipiens f. molestus could be distinguished from Cx. quinquefasciatus and intermediates by characters of the female wing (intersection of the subcostal vein with the costal in relation to the level of furcation of R1 + 2), abdominal terga, male scutum, and phallosomes.Krtinic et al. (2015) found significant differences in the number of pecten spines and siphonal index between biotypes (pipiens and molestus) for both sexes.Thus, morphological differentiation based on wing shape aids in taxonomic analyses of biotypes in the Cx.pipiens complex, especially in areas with potential for WNV expansion in South America (Figueroa et al. 2020).
This study aims to compare continental (from 27° S to 41° S latitude) and insular populations of Cx. pipiens s.s.using GM methods.Specifically, we compare wing size and wing shape and use these traits to evaluate differences between and within biotypes.Geographic isolation is an important factor in driving differences in wing shape among Aedes albifasciatus Macquart populations from the 3 ecoregions of Argentina (Garzón and Schweigmann 2018); thus, geographic isolation could determine differences in wing shape between the Cx.pipiens s.s. and its biotypes along continental and insular sites in South America.

Collection and Processing
Larvae of Cx. pipiens s.s. were collected from artificial habitats, mainly urban and suburban cemeteries, located between 27° S and 41° S, and from seminatural ponds (Table 1).Cemetery vases were monitored until reaching at least 100 individuals per locality (about 3-6 vases).Adults were collected from underground and aboveground habitats in rural areas using a hand-held vacuum cleaner.All larvae collected were transported in containers with water from the site where they were collected.Then larvae were transferred to 300 cc plastic containers (randomly 50 larvae per container) prepared with demineralized water and fish food was added daily.Emergent adults were transferred using a mouth aspirator, killed in a deep freezer, and identified.Culex pipiens complex specimens were identified under a stereomicroscope using the identification keys of Rossi and Almirón (2004), and González et al. (2015, 2016).

Molecular Analysis
Once the sex of each specimen was determined and the wings removed, the whole body was used to extract DNA with the method described by Aljanabi and Martinez (1997).The protocols of Smith and Fonseca (2004), and Bahnck and Fonseca (2006) were used to distinguish Cx. pipiens s.s.from Cx. quinquefasciatus and to identify biotypes and hybrids within Cx. pipiens s.s., respectively.
Once Cx. pipiens s.s. was confirmed, the CQ11 locus was amplified to differentiate biotypes.The PCR were carried out according to Bahnck and Fonseca (2006), using the pipCQ11R, molCQ11R, and CQ11F primers.The PCR assay used was standardized for a 20-µl reaction volume.Reactions contained 0.12 μl Taq, 0.5 μl BSA, 0.5 μl MgCl 2 , 2.4 μl dNTP, 1.3 μl buffer, 0.5 μl primer pipCQ11R for biotype pipiens, 0.75 μl primer molCQ11R for biotype molestus, and 0.75 μl primer CQ11F, 2 μl genomic DNA, and 11.2 μl free water.The PCR was performed with an initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 40 s, with a final extension at 72 °C for 5 min.Amplified fragments were visualized on 1% agarose gel: a single DNA fragment of 200 bp corresponds to the pipiens biotype, a single DNA fragment of 250 bp corresponds to the molestus biotype and individuals presenting both fragments were considered as hybrids.

Geometric Morphometrics Analysis
The right wing of each specimen was removed and mounted a glass microscope slide with a 0.08-to 0.12-mm glass coverslip.Culex wings were then photographed using a Stemi 508 Zeiss digital camera connected to a stereomicroscope with the Axiocam program.A total of 23 landmarks were digitized on the wings using the software TPSUtil v1.58 and TPSDig v2.17 (Rohlf 2015).Landmarks along the main veins were chosen, so that the configurations were repeatable, appropriate for each wing, and to cover the wing shape as completely as possible (Fig. 1).The landmark selection was consistent with other studies analyzing mosquito wing morphometrics (Lorenz et al. 2017, Martinet et al. 2021).
Multivariate analyses were performed using MorphoJ v. 1.07a (Klingenberg 2011) and the R program (R Core Team 2016).The raw landmark coordinates for all individuals were aligned and superimposed in MorphoJ, using the Procrustes superimposition function to remove variation due to differences in scale, position, and orientation from the coordinates (Rohlf and Slice 1990).Centroid size is defined as the square root of the sum of the squared distances between the centroid and each landmark and can be used as a proxy for wing size (Bookstein 1997).So, the centroid size and Procrustes coordinates obtained from landmark data were used for further statistical analyses.
Wing size and shape were analyzed between biotypes (molestus, pipiens, and hybrids) and between geographic areas: northern area (27°S-29°S), central area (sites located around 33°S), southern area (36°S to 41°S), and island (Rapa Nui), using the same set of individuals.Environmental and climatic conditions are similar within each of these areas (Sánchez and Morales 2002).
To visualize the relative morphological changes between populations, a warped outline drawing was made in TPSDig2 (Rohlf 2015), which will be used to visualize the mean shape of Culex specimens.Then a covariance matrix of individuals was created in R using libraries geomorph, Morpho, plyr, dplyr, and ggplot2, to perform a principal component analysis (PCA) and an average PCA of each biotype and geographic area, and canonical variate analysis (CVA).CVA was performed through 10,000 permutations of Mahalanobis distance from the pooled within-location covariance matrix.The mean shape of Cx. pipiens s.s. and biotypes per geographic area were extracted, and the covariance matrix of mean shape variation was used to identify graphically morphological variation between geographic areas and biotypes.The allometric influence of wing size on wing shape was assessed by multivariate regression of the Procrustes coordinates against centroid size, using a permutation test with 10,000 randomizations (Monteiro 1999).A comparative analysis of the centroid sizes was performed for populations of both sexes independently using a Procrustes analysis of variance (ANOVA), with 1,000 permutations using procD.lm (geomorph function).

Molecular Analysis
Locus ace-2 showed a 600 bp DNA fragment for all individuals; thus, 100% of samples were identified as Cx.pipiens s.s.Sequences of the COI gene obtained from some individuals were contrasted with these in GenBank; we confirmed Cx. pipiens in all individuals analyzed.The microsatellite CQ11 was successfully amplified in 153 of the 194 specimens studied.This locus showed the presence of both alleles (200 and 250 bp) and both homozygotes and the heterozygote in the samples.Of these, 58.8% were identified as Cx.pipiens f. molestus, 11.8% were Cx.pipiens f. pipiens, and 29.4% were classified as hybrids between the two.
Biotype molestus was present in all collection sites, while biotype pipiens was present in 5 of the 10 localities (Fig. 2).Notably, biotype pipiens was observed in the coastal localities (<10 km from the coast).However, only biotype molestus was identified in Rapa Nui.Biotype pipiens was not found underground in Melipilla (ME) or the sampling sites of Copiapó (CC and TB).Adults from Trehuaco were all females collected after biting in a rural house; 2 of the 18 specimens were described as Cx.pipiens f. pipiens.

Geometric Morphometrics Analyses
The proportion of shape variance that was explained by size (allometry) was found to be 4.44% and 7.65% in the male and female, respectively.This shows an effect of size on shape variance.ANOVA showed a significant wing shape effect related to sexual dimorphism (F 42 = 93.19,P < 0.0001), which was not determined by allometry.Analysis was carried out separately females and males.

Analysis by Biotypes
In the CVA, the wing shape of females of Cx. pipiens s.s.differed among the 3 biotypes (H: hybrids, M: molestus, and P: pipiens) (Fig. 3A).The wing shape of the 3 biotypes in males was clearly segregated in the first 2 canonical axes, with more similarity between the wing shape of hybrids, and molestus individuals compared to pipiens (Fig. 3B).Procrustes ANOVA showed differences in the centroid size between biotypes in females (F 3 = 3.15, P = 0.0276) and males (F 126 = 2.15, P = 0.0184), but not in wing shape (Table 2).However, only individuals with biotype molestus from Rapa Nui showed shorter wings than the other biotypes, notorious in males (Fig. 4).The main differences between biotypes of the sexes of Cx. pipiens s.s. were associated with landmarks 1 (interception subcostal vein with costal vein) and 15 (furcation of R 2 + 3).

Analysis by Geographic Area
The first 2 components of the PCA accumulated 53.04% of the total variation of shape in females (PC1: 32.26% and PC2: 20.78%) (Fig. 5A) and 47.22% in males (PC1: 29.45% and PC2: 17.77%) (Fig. 5B).Male populations were clearly segregated, while female populations overlapped.Males from Rapa Nui showed a different wing shape than the mainland sites.In fact, males from Rapa Nui showed more compressed and smaller wings than those from the other sites, as indicated by the smaller distance between LM 16 and LM19.Procrustes ANOVA showed significant differences among geographic areas in centroid size and wing shape in both females and males (Table 3).
The main relative changes between geographical areas were determined by LMs 1 (interception of subcostal vein with costal vein), 15 (furcation of R 2 + R 3 ), and 16 (furcation of M 1 + M 2 ).Major changes were observed in these landmarks in females and males.In the island sites, the subcostal intersected the costal before the furcation of R 2 + R 3 , whereas at the other sites, the subcostal intersected the costal beyond the furcation of R 2 + R 3 .Females and males of Rapa Nui have shorter wings than the continental individuals.
The wing shape of females (Fig. 6A) and males in the CVA (Fig. 6B) differed among the 4 geographic areas (N: North, C: Central, S: South, and I: Island).The analysis showed a gradual differentiation among the continental sites in females, and all these sites differed in relation to the island.This pattern was also observed in the wings of males, and the shape of those from the island showed more differences compared to the other sites, followed by the individuals located in the central area.
The centroid size indicated that most mosquitoes from Rapa Nui had shorter wings than continental individuals (North, Central, and South populations), being more evident in males (Fig. 7).

Discussion
The results of this study showed clear geographic variation in wing shape and morphology in Cx. pipiens s.s.Both biotypes and hybrids     were recorded in all continental sites studied.The CQ11 assay amplified 78.8% of the specimens studied, identifying 58.8% of these as biotype molestus, 11.8% as biotype pipiens, and 29.4% as hybrids.
Both ecological forms of Cx. pipiens s.s. were found in sampling sites sharing the larval habitat and in both rural and urban habitats.The biotype molestus was found in an underground site and in aboveground sites, especially in a seminatural pond in Copiapó (TB).However, biotype pipiens was found only in aboveground sites, especially cemetery vases.This agrees with Vogels et al. (2016), who found Cx. pipiens f. pipiens and Cx.pipiens f. molestus in sympatry in aboveground locations.
Females from the Trehuaco site were collected after biting humans in a rural home, and 2 of the 18 specimens were described as Cx.pipiens f. pipiens.In the Northern Hemisphere, females of biotype pipiens primarily feed on birds and are therefore important vectors of pathogens circulating in bird populations to humans (Haba and Mcbride 2022).
Interestingly, relatively few larval habitats with biotype pipiens were identified, which could be associated with the number of samples in each area.On Rapa Nui, about 3,600 km west of the Chilean coast, all individuals were classified as biotype molestus.From its origin in the Mediterranean (according to the best supported hypothesis) Cx. pipiens f. molestus have spread significantly through human intervention, establishing worldwide distribution (Aardema et al. 2022) and reaching remote islands such as Rapa Nui.The first records of Cx. pipiens s.s. in Rapa Nui Island date back to the early 1900s (Fuentes 1914); its introduction was probably due to human transport from the American continent, as with the dengue vector Aedes aegypti Linnaeus, another mosquito present in Rapa Nui.
Culex pipiens s.s. and related species are probably the best studied examples of hybridization in mosquitoes.Hybridization is an intrinsically ecological process that can significantly influence different traits of individuals, such as response to abiotic environmental conditions, and as a biotic interaction, hybridization contributes to shaping patterns of biodiversity, and its spatial structure in species and communities (Porretta and Canestrelli 2023).Actual hybridization between mosquito species has been widely observed and elegantly demonstrated genetically (Savage and Kothera 2012).We found hybrids at all continental sites, mainly sharing larval habitats with the biotype molestus.
Diapause in Cx. pipiens s.s. has been well described (Robich andDenlinger 2005, Meuti et al. 2015).In the continental sites analyzed in this present study, Cx. pipiens f. pipiens was observed under different climatic conditions (summer and winter), ranging from an arid climate in the north to a temperate humid climate in the south.Therefore, specific studies of the diapause process will be necessary to understand the physiological requirements of Cx. pipiens s.s. in this part of its distribution.
Morphometric analysis of the biotypes showed significant differences only in wing centroid size for both females and males.Although we did not find significant differences in wing shape, it was possible to infer that some individuals of biotype molestus had shorter wings than biotype pipiens and hybrids, but these individuals were from Rapa Nui.Therefore, the differences observed in biotypes are associated with geographic area, and consequently, geographic isolation could be the principal driver that determines wing size in Cx. pipiens s.s. in South America.
To our knowledge, Krtinic et al. (2015) performed the first analysis assessing differences in wing shape between biotypes, describing differences in wing centroid size between females and males.A pairwise comparison showed that the wing centroid size of the hypogeous biotype was significantly smaller for both sexes compared to the epigeous biotypes.Other morphological characters, such as the siphonal index, have been commonly used to determine the taxonomic status of Cx. pipiens s.s.biotypes (Vinogradova 2003, Krtinić et al. 2015).However, an integrative approach including massive sequencing is necessary to clarify the taxonomic determination of the Cx.pipiens complex.
Procrustes ANOVA showed significant differences in centroid size and wing shape between the geographic areas studied.Individuals from the central and northern areas differed more, and all these locations differed from the island.In particular, the Rapa Nui population had shorter wings than continental populations.
The wing size and shape of insects are largely influenced by environmental factors such as temperature, relative humidity, larval density, and food availability (Morales-Vargas et al. 2010, Krtinić et al. 2015, Dellicour et al. 2017).However, since its introduction to the island, Cx. pipiens s.s.appears to have phenotypic variation related to geographic isolation, as described in Pantala flavescens Fabricius, another cosmopolitan insect present on the island (Alvial et al. 2019).The smaller wings observed in the insular population of this mosquito can be associated with the "island flightlessness hypothesis," which is explained if a new species colonizing an isolated island is subject to a strong selective force against dispersal because flight could be disadvantageous in that the energy used to maintain and use the flight muscles is effectively wasted (Whittaker and Fernandez-Palacios 2007).Smaller wings might also indicate a reduction in body size ("island rule theory"), where body size is selected toward an optimal body size at which organisms maximize their reproductive output (Lokatis and Jeschke 2018).However, future studies on these topics are needed to confirm that this occurrence is greater than expected by chance alone.
In all comparative analyses, we found that the male populations were more spatially structured than the female populations.This pattern of sexual dimorphism has been previously recognized in culicid species (Virginio et al. 2015).Male mosquitoes do not require blood for reproduction or survival; they are completely dependent on sugars and the reserves they have acquired from larval feeding, in addition to adult food supply (Wilkerson et al. 2021).Therefore, food availability is critical for larval development.Some species produce smaller adults when food is limited (Carvajal-Lago et al. 2021).However, Cx. pipiens s.s.individuals were found in a microenvironment with an abundance of nutrients, so survival would not be dependent on nutrient availability.In addition, if larvae live in habitats that are too polluted, temporary or inaccessible, predation may not be important.Therefore, the geographic patterns observed in this analysis, mainly in male populations, may be determined by local factors, such as food availability and proximity to water bodies.
Several studies of culicids have shown that wing shape is an indicator of microevolution.Although most quantitative genetic information cannot be obtained from wings (Dujardin 2008), morphometric analyses of wing size and shape are suitable and can serve as a preliminary assessment of evolutionary patterns in culicids (Lorenz et al. 2017).Two and 4 yr were sufficient for Ae.albopictus and Ae.aegypti, respectively, to accumulate wing shape changes (Vidal et al. 2012, Louise et al. 2015).Therefore, the 100 yr that Cx. pipiens s.s.have been on Rapa Nui may be sufficient to indicate microevolutionary processes as a result of its extreme isolation.
The differences found using geometric morphometrics tools between biotypes and geographical areas in Cx. pipiens s.s.provide new evidence of the effects of geographic and environmental processes on mosquitoes of health importance.

Fig. 1 .
Fig. 1.Twenty-three landmarks on the wing of an Culex pipiens s.l.mosquito for GM analyses.

Fig. 2 .Fig. 3 .
Fig. 2. Composition of Culex pipiens genotypes from ten field-collected populations in Chile using the CQ11 microsatellite assay.The nine continental sites and one insular site (Rapa Nui) are indicated by blacks dots.

Table 1 .
Collection ofCx.pipiens s.s.mosquitoes identified and used for molecular and morphometric analysis.Sites are grouped by geographic area (N: north, C: central, S: south, and I: island)

Table 2 .
Procrustes ANOVA model of shape among biotypes for females (a) and males (b) MS-mean sum of squares, SS-sum of squares, *significant p values.

Table 3 .
Procrustes ANOVA model of shape variable among geographic area for females (a) and males (b) MS-mean sum of squares, SS-sum of squares, *significant p values.